Nerve repair scaffolds having high microchannel volume and methods for making the same

ABSTRACT

Tissue scaffolds for neural tissue growth have a plurality of microchannels disposed within a sheath. Each microchannel comprises a porous wall having a thickness of ≤about 100 μm that is formed from a biocompatible and biodegradable material comprising a polyester polymer. The polyester polymer may be polycaprolactone, poly(lactic-co-glycolic acid) polymer, and combinations thereof. The tissue scaffolds have high open volume % enabling superior (linear and high fidelity) neural tissue growth, while minimizing inflammation near the site of implantation in vivo. In other aspects, methods of making such tissue scaffolds are provided. Such a method may include mixing a reduced particle size porogen with a polymeric precursor solution. The material is cast onto a template and then can be processed, including assembly in a sheath and removal of the porogen, to form a tissue scaffold having a plurality of porous microchannels.

GOVERNMENT SUPPORT

This invention was made with government support under EB014986 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application under 35 U.S.C.371 of International Application No. PCT/US2016/056104 filed on Oct. 7,2016 and published in English as WO 2017/062845 A1on Apr. 13, 2017. Thisapplication claims the benefit and priority of U.S. Application Ser. No.62/238,506 filed on Oct. 7, 2015. The entire disclosures of the aboveapplications are incorporated herein by reference.

FIELD

The present disclosure relates to tissue scaffolds incorporating porousmicrochannels to promote neural tissue growth and methods for makingsuch tissue scaffolds.

BACKGROUND

Although the peripheral nervous system (PNS) has a greater capacity forregeneration than the central nervous system (CNS), functionalregeneration after injury is largely incomplete if injured axons becomemisaligned or lose contact with innervated tissues. Major functionaldeficits result and include deficient re-innervation of target tissuesand painful neuroma formation.

Factors that influence PNS regeneration include the nature and the levelof the damage itself, the period of denervation, the type and diameterof the damaged nerve fibers, and age. Proximal nerve injuries orcomplete transection of a large gap of the nerve generally have pooreroutcomes with minimal clinically meaningful motor and sensory recovery.Several reasons contributing to suboptimal recovery have been identifiedand include: 1) deficiencies in rate of axonal regrowth; 2) compromiseto an otherwise permissive environment for axonal elongation; 3) changesin the target tissue or path to reach the target tissue; 4) excessiveand chronic neuroinflammation; and 5) Schwann cell (SC) atrophy anddysfunction.

Currently, the standard in clinical practice for surgical repair ofperipheral nerve interface (PNI), in which there is a large gap in theperipheral nerve, involves placement of autologous nerve grafts.Disadvantages of autografts include: 1) donor site morbidity; 2) limitedsupply of donor grafts; and 3) increased time and complexity of surgery.

Experimental development of scaffolds to support peripheral nerve repairhave resulted in commercially available nerve guides, but thesescaffolds provide only single large diameter tubes that result inmisalignment of regenerating axons with their proper targets. In oneexample, NEUROGEN™ sold by Integra LifeSciences is an open tubescaffold. Upon implantation with a transected rat sciatic nerve model,such an open tube scaffold shows that many axons undesirably lose linearorientation along a proximal end, only 200 μm after they enter thescaffold, prior to reaching the other distal end. Axons are less denseand of those that reach the distal end, some still lose orientation evenas they exit into the distal nerve. This misguidance of axons can causepain due to neuroma. Furthermore, such commercially available scaffoldslack seeding with growth-promoting substances, such as growth factors.Recently, cellular approaches including development of conduits filledwith Schwann cells have shown some success because Schwann cellsnaturally support axonal regeneration by guiding and supporting axongrowth, but these cells have not been translated for human peripheralnerve injury.

Moreover, there are no effective therapies for promoting regenerationafter either acute or chronic spinal cord injuries (SCI) in humans.Various experimental approaches promote axonal regeneration in SCIanimal models, including cell grafting to sites of injury to supportaxonal attachment and elongation. Grafted cells include astrocytes,Schwann cells, marrow stromal cells or stem cells. However, a drawbackof cellular implants is a lack of 3D organization, resulting in randomdirections of axon growth; most axons do not regenerate beyond theinjury site into host tissue, and hence functional recovery is extremelymodest if present at all.

Thus, there remains a need to identify strategies and technologies forenhancing the extent, rate, guidance, targeting and lesion-distance overwhich neural tissue (e.g., axons) can regenerate.

SUMMARY

This section provides a general summary of the disclosure, and is not acomprehensive disclosure of its full scope or all of its features.

In certain aspects, the present disclosure provides new tissue scaffoldsfor neural tissue growth. In one aspect, the disclosure provides such atissue scaffold that comprises a plurality of microchannels disposedwithin a sheath. Each microchannel comprises a porous wall having athickness of less than or equal to about 100 μm. The porous wallcomprises a biocompatible and biodegradable material comprising apolyester polymer. The polyester polymer may be selected from a groupconsisting of: polycaprolactone, poly(lactic-co-glycolic acid) polymer,and combinations thereof.

In other aspects, the present disclosure provides methods of making atissue scaffold for promoting neural tissue growth. Such a method maycomprise admixing a porogen with a polymeric precursor solution to forma suspension. The porogen has an average particle size of less than orequal to about 40 μm. The polymeric precursor solution comprises abiocompatible and biodegradable polyester polymer precursor and a firstsolvent. Then, a template is contacted with the suspension to coat atleast one surface. At least a portion of the first solvent is volatizedfrom the material on the template to form a coating. The coating is thenremoved from the template. The porous microchannel may be disposed orassembled inside a sheath with a plurality of other porousmicrochannels. Finally, the porogen may be removed to form a porousmicrochannel and thus, the tissue scaffold is formed having a pluralityof porous microchannels arranged therein.

Further areas of applicability will become apparent from the descriptionprovided herein. The description and specific examples in this summaryare intended for purposes of illustration only and are not intended tolimit the scope of the present disclosure.

DRAWINGS

The drawings described herein are for illustrative purposes only ofselected embodiments and not all possible implementations, and are notintended to limit the scope of the present disclosure.

FIG. 1 shows a perspective view of an exemplary implantable tissuescaffold device having a plurality of porous microchannel tubescontained with a sheath according to certain aspects of the presentdisclosure.

FIGS. 2A-2C. FIG. 2A shows a picture of a cross-section of an ulnarnerve showing natural axonal architecture. FIGS. 2B-2C showcross-sectional schematics of two different high lumen volume nerverepair scaffolds which ideally match or emulate a nerve's nativearchitecture according to certain aspects of the present disclosure. Thedesign in FIG. 2C has a higher microchannel density than a microchanneldensity in FIG. 2B.

FIG. 3 shows a schematic of a scaffold fabrication process according tocertain aspects of the present disclosure.

FIGS. 4A-4D. FIG. 4A shows a fabricated microchannel. FIG. 4B shows afabricated outer sheath. FIG. 4C shows an assembly device with a customclaim shell slot die for assembling microchannels within a sheath. FIG.4D shows the device inserting a microchannel array into an outer sheath.

FIGS. 5A-5C. FIGS. 5A-5C are pictures of a porogen salt-templatedpolycaprolactone (PCL) scaffold. FIG. 5A shows one terminal end of thescaffold, while FIG. 5B is a magnified view taken from the rectangleindicated in FIG. 5A showing the microchannels (having a scale bar of500 μm). FIG. 5C is a side view of the tissue scaffold in FIGS. 5A-5B.

FIG. 6 shows a cross-sectional SEM image of a 30 vol. % PCL-70 vol. %NaCl porogen material before salt leaching (immersion in water to removethe porogen); fracture surface. An average NaCl particle size is about17 μm prior to salt leaching.

FIGS. 7A-7B show SEM fracture surface images of PCL. FIG. 7A shows PCLwithout any porogen salt-templated that is therefore 100% dense. Thescale bar is 2 μm. FIG. 7B shows porogen salt-templated PCL, exhibitingsubstantial porosity created by NaCl particles. The scale bar is 5 μm.

FIGS. 8A-8D. FIGS. 8A-8D are SEM images of porogen salt-templated PLGA85/15. FIG. 8A shows 0% porosity (scale bar is 5 μm), FIG. 8B shows 40%porosity (scale bar is 20 μm), FIG. 8C shows 50% porosity (scale bar is10 μm), and FIG. 8D shows 60% porosity (scale bar is 20 μm).

FIG. 9 shows Young's modulus of PCL as a function of PCL volume % (n=5for each % porosity).

FIGS. 10A-10C. FIGS. 10A-10C show a hybrid micro-drilled agarosemicrochannel scaffold. FIG. 10A shows a terminal end view of themicrochannel scaffold (scale bar is 300 μm) and FIG. 10B shows a sideview of the microchannel scaffold (scale bar is 1 mm). A porogensalt-templated PCL sheath is shown in FIG. 10C (scale bar is 1 mm).

FIG. 11 is a photograph taken of an implanted hybrid PCL sheath andagarose scaffold (shown in FIGS. 10A-10C) after 8 weeks in vivo. Thescaffold remains intact with no noticeable sign of inflammation.

FIGS. 12A-12B. After 8 weeks in vivo, the implanted hybrid sheath andscaffold (shown in FIGS. 10A-10C) show neurofilament growth (in green)and supporting neural cells (Schwann cells). As can be seen, robust axonand Schwann cell integration occurs and ingress (proximal) and egress(distal) is apparent from the agarose microchannels.

FIGS. 13A-13C. Tissue scaffolds have chitosan sheaths housing amicro-drilled agarose scaffold. FIG. 13A shows a picture of a chitosansheath and agarose scaffold adjacent to a penny coin. The chitosancaused significant inflammation despite the use of low endotoxin gradechitosan. FIG. 13B is an optical image showing the agarose scaffold(linear features are the intact channels) is intact at the experimentcompletion. FIG. 13C shows Nissl staining demonstrating significantinflammation (yellow arrows) along the scaffold periphery.

FIGS. 14A-14C. FIGS. 14A-14C show tissue growth in a tissue scaffoldhaving a PCL sheath and PCL microchannel scaffold according to certainaspects of the present disclosure implanted in a rat sciatic nerve 4weeks post implantation (left side is proximal, right side is distal).FIG. 14A shows a cross-section stained with S100 to highlight Schwanncells. FIG. 14B shows a cross-section stained with NF200 to highlightneural tissue. FIG. 14C shows a magnified view of the distal end of FIG.14B highlighting axons penetrating into host tissue after egress fromthe scaffold.

FIG. 15 shows a tissue scaffold having a PCL sheath and an array of PCLmicrochannels for use as a CNS scaffold. The scale bar is 300micrometers.

FIG. 16 is a cross-section showing a CNS tissue scaffold having a PCLsheath and an array of PCL microchannels implanted in a rat T3 fulltransection. “W” and “C” are abbreviations for scaffold walls andchannels respectively. NF200 stain highlights axons in white; yellowarrows point to regenerated axons. Scale bar is 500 micrometers.

FIGS. 17A-17C. FIGS. 17A-17C show cell attachment on a control and PCLmaterials for purposes of comparison. 3T3 fibroblast cells are stainedfor actin and nucleus. FIG. 17A shows a positive control of cell growthin a well plate. FIG. 17B shows cell growth on non-porous PCL (100% byvolume PCL). FIG. 17C shows 3T3 fibroblast cell growth a porous PCLprepared in accordance with certain aspects of the present disclosurehaving 30 volume % PCL (70 volume % porosity). All scale bars are 100μm.

FIGS. 18A-18C. FIGS. 18A-18C show that the walls of microchannels haveinterconnected porosity. FIG. 18A shows an SEM of the surface of theinner wall, FIG. 18B shows an SEM of the cross-section of the wall, andFIG. 18C the outer surface of the wall of the microchannel. All scalebars are 4 μm.

FIGS. 19A-19B. FIG. 19A is rat spinal cord tissue scaffold having over85% open volume prepared according to certain aspects of the presentdisclosure (scale bar is 300 μm). FIG. 19B is a pig sciatic nervescaffold having over 85% open volume prepared according to certainaspects of the present disclosure (scale bar is 4 mm).

FIGS. 20A-20E. FIG. 20A (including FIGS. 20A(1)-20A(4)) include stainsshowing scaffold performance in a rat transected sciatic nerve. FIGS.20A(1)-20A(2) show distal and proximal ends of a multichannel 10 mm PCLscaffold prepared in accordance with certain aspects of the presentdisclosure in transected rat sciatic nerve, 4 weeks post implant. Axonsare labeled in red using NEUROFILLAMENT™ 200. Arrow heads point wherethe proximal transected nerve stump is anastomosed to the scaffold. Fullarrows point to linear axons in the channels on the proximal and distalparts of the scaffold as well as in the egress. FIGS. 20A(3)-20A(4) showa conventional scaffold (NEURAGEN™) at the same time frame in atransected rat sciatic nerve. The axons have loose orientation shortlyafter they enter the scaffold (full arrows and circle where axons areperpendicular to regeneration axis). The few that reach the distal sideare not oriented even if they exit to the egress. The scale bars are 200μm. FIG. 20B shows tissue growth in a 15 mm agarose scaffold loaded withBDNF secreting-marrow stromal cells with additional distal nerveinjection of BDNF to attract regenerating axons to exit the scaffoldinto the distal nerve. FIG. 20C (including FIGS. 20C(1)-20C(2)) showproximal and distal ends with many myelinated axons bridges in BDNFtreated animals. FIG. 20C(1) is red—NF200. FIG. 20C(2) is green—S100.FIG. 20D (including FIGS. 20D(1)-20D(2)) are SEMs of the channelsshowing many myelinated axons in the BDNF treatment, similar to theautograft as well as vascularization (asterisks). FIG. 20E showsquantification of axon density 12 mm within the scaffold indicating thatin BDNF group axon density in channel core is similar to syngeneic nerveautografts. **P<0.05 (comparing scaffold group loaded withGFP-expressing cells to other groups).

FIGS. 21A-21C. FIG. 21A is a schematic of nerve stump apposed with thescaffold interface inside the overhang sheath. The epineurium is thensutured to the overhang (arrows). The sleeve can be seen in agarosehydrogel scaffold (FIG. 21B) and folded out (white arrow) in PCLscaffold (FIG. 21C).

FIGS. 22A-22B. FIG. 22A shows a schematic of porcine surgery site (arrowindicating surgical site). FIG. 22B shows a 15 mm scaffold, fabricatedwith PCL, implanted in transected porcine sciatic nerve. White arrowspoint to the suture where the scaffold is anastomosed with theepineurium of the nerve stumps.

FIGS. 23A-23D. FIGS. 23A-23D show a scaffold 3 months post implantationin for a transected pig sciatic nerve site. FIG. 23A shows a siteoverview in a horizontal plane. Many Schwann cells (S100—red) areevident on the proximal side. Scale is 2 mm. FIGS. 23B-23C have axonlabeling (NF200—green) showing axonal penetration into the scaffold onthe proximal side. These reach the distal side of the scaffold in alinear fashion. White arrows point to the channel, scale is 100 μm. FIG.23D shows that associated axons and Schwann cells are observed in thedistal stump, 4 mm beyond the distal end of the scaffold. Scale is 20μm.

FIGS. 24A-24C. FIG. 24A (including FIGS. 24A(1)-24A(3)) shows a skinsensory test just after sciatic nerve transection in a pig. FIG. 24B(including FIGS. 24B(1)-24B(2)) shows preliminary results 3 months postscaffold implant. FIG. 24C shows the area on the skin is innervated bythe Common fibular nerve—a branch of the sciatic nerve.

FIGS. 25A-25C. FIG. 25A is a schematic of the anatomical structurespresent in a representative peripheral nerve structure. FIG. 25B shows across-sectional view of a terminal end of a micro-scaffold prepared inaccordance with certain aspects of the present disclosure designed tomimic the natural architecture of a peripheral nerve. FIG. 25C showscommercially available, Federal Drug Administration (FDA)-approvedhollow tube scaffold devices from left to right: NEURAGEN™ nerve guideavailable from Integra Lifesciences Corp., NEUROTUBE™ available fromSynovis Microcompanies Alliance, and NEUROLAC™ available fromPolyganics.

FIGS. 26A-26C. FIGS. 26A-26C show comparative nerve guide implants in arat transected sciatic nerve, 4 weeks post implantation. The axons arestained with Green-NF200. FIG. 26A shows the commercially availableNEURAGEN™ nerve guide. FIG. 26B shows a microchannel scaffold deviceprepared in accordance with certain aspects of the present disclosure.FIG. 26C shows an autograft. The interrupted lines demarcate theimplant-nerve interface. The arrows on the left point to the proximalside of the implant, while the arrows on the right point to the distalaspect of the implant.

FIG. 27 shows high magnification view of different areas from FIGS.26A-26C of the implanted comparative nerve guide devices (NEURAGEN™nerve guide, a microchannel scaffold device prepared in accordance withcertain aspects of the present disclosure, and an autograft) for a rattransected sciatic nerve, 4 weeks post implantation. Views of stainedaxon growth are shown at the nerve stump, proximal scaffold,mid-scaffold, distal scaffold, distal nerve, and end of block. ScaleBar: 100 μm.

FIG. 28 shows quantification of regenerating axons in different parts ofimplanted comparative nerve guide devices (NEURAGEN™ nerve guide, amicrochannel scaffold device prepared in accordance with certain aspectsof the present disclosure, and an autograft) from proximal to distalnerve for a rat transected sciatic nerve, 4 weeks post implantation.ANOVA, mean±S.E.M, p<0.001.

FIG. 29 shows pinprick scores after 7 weeks of implantation forcomparative devices, including a PCL scaffold prepared in accordancewith certain aspects of the present disclosure (N=3 rats/group) and aNeuragen™ nerve guide device (compared to a medial nerve). The ratshaving a porous PCL rats demonstrate earlier and accelerated sensoryrecovery compared to NeuraGen® implanted rats after injury.

FIGS. 30A-30D. A microchannel scaffold device prepared in accordancewith certain aspects of the present disclosure is shown implanted in pigtransected sciatic nerve, 4 months post-surgery. FIG. 30A shows a 15 mmdevice, fabricated with PCL, implanted in transected porcine sciaticnerve. Arrows point to a suture where the device is anastomosed with theepineurium of the nerve stumps. FIGS. 30B and 30C show axon labelingwith NF200—green. FIG. 30B shows axonal penetration into the device onthe proximal side, while FIG. 30C shows axons reaching the distal sideof the device in a linear fashion. The interrupted line demarcateshost-scaffold interface. FIG. 30D shows associated axons and Schwanncells observed in the distal stump, 3.5 mm beyond the distal end of thedevice. Scale in FIGS. 30B-30C: 50 micrometers.

Corresponding reference numerals indicate corresponding parts throughoutthe several views of the drawings.

DETAILED DESCRIPTION

Example embodiments are provided so that this disclosure will bethorough, and will fully convey the scope to those who are skilled inthe art. Numerous specific details are set forth such as examples ofspecific compositions, components, devices, and methods, to provide athorough understanding of embodiments of the present disclosure. It willbe apparent to those skilled in the art that specific details need notbe employed, that example embodiments may be embodied in many differentforms and that neither should be construed to limit the scope of thedisclosure. In some example embodiments, well-known processes,well-known device structures, and well-known technologies are notdescribed in detail.

The terminology used herein is for the purpose of describing particularexample embodiments only and is not intended to be limiting. As usedherein, the singular forms “a,” “an,” and “the” may be intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. The terms “comprises,” “comprising,” “including,” and“having,” are inclusive and therefore specify the presence of statedfeatures, elements, compositions, steps, integers, operations, and/orcomponents, but do not preclude the presence or addition of one or moreother features, integers, steps, operations, elements, components,and/or groups thereof. Although the open-ended term “comprising,” is tobe understood as a non-restrictive term used to describe and claimvarious embodiments set forth herein, in certain aspects, the term mayalternatively be understood to instead be a more limiting andrestrictive term, such as “consisting of” or “consisting essentiallyof.” Thus, for any given embodiment reciting compositions, materials,components, elements, features, integers, operations, and/or processsteps, the present disclosure also specifically includes embodimentsconsisting of, or consisting essentially of, such recited compositions,materials, components, elements, features, integers, operations, and/orprocess steps. In the case of “consisting of,” the alternativeembodiment excludes any additional compositions, materials, components,elements, features, integers, operations, and/or process steps, while inthe case of “consisting essentially of,” any additional compositions,materials, components, elements, features, integers, operations, and/orprocess steps that materially affect the basic and novel characteristicsare excluded from such an embodiment, but any compositions, materials,components, elements, features, integers, operations, and/or processsteps that do not materially affect the basic and novel characteristicscan be included in the embodiment.

Any method steps, processes, and operations described herein are not tobe construed as necessarily requiring their performance in theparticular order discussed or illustrated, unless specificallyidentified as an order of performance. It is also to be understood thatadditional or alternative steps may be employed, unless otherwiseindicated.

When a component, element, or layer is referred to as being “on,”“engaged to,” “connected to,” or “coupled to” another element or layer,it may be directly on, engaged, connected or coupled to the othercomponent, element, or layer, or intervening elements or layers may bepresent. In contrast, when an element is referred to as being “directlyon,” “directly engaged to,” “directly connected to,” or “directlycoupled to” another element or layer, there may be no interveningelements or layers present. Other words used to describe therelationship between elements should be interpreted in a like fashion(e.g., “between” versus “directly between,” “adjacent” versus “directlyadjacent,” etc.). As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items.

Although the terms first, second, third, etc. may be used herein todescribe various steps, elements, components, regions, layers and/orsections, these steps, elements, components, regions, layers and/orsections should not be limited by these terms, unless otherwiseindicated. These terms may be only used to distinguish one step,element, component, region, layer or section from another step, element,component, region, layer or section. Terms such as “first,” “second,”and other numerical terms when used herein do not imply a sequence ororder unless clearly indicated by the context. Thus, a first step,element, component, region, layer or section discussed below could betermed a second step, element, component, region, layer or sectionwithout departing from the teachings of the example embodiments.

Spatially or temporally relative terms, such as “before,” “after,”“inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and thelike, may be used herein for ease of description to describe one elementor feature's relationship to another element(s) or feature(s) asillustrated in the figures. Spatially or temporally relative terms maybe intended to encompass different orientations of the device or systemin use or operation in addition to the orientation depicted in thefigures.

Throughout this disclosure, the numerical values represent approximatemeasures or limits to ranges to encompass minor deviations from thegiven values and embodiments having about the value mentioned as well asthose having exactly the value mentioned. Other than in the workingexamples provided at the end of the detailed description, all numericalvalues of parameters (e.g., of quantities or conditions) in thisspecification, including the appended claims, are to be understood asbeing modified in all instances by the term “about” whether or not“about” actually appears before the numerical value. “About” indicatesthat the stated numerical value allows some slight imprecision (withsome approach to exactness in the value; approximately or reasonablyclose to the value; nearly). If the imprecision provided by “about” isnot otherwise understood in the art with this ordinary meaning, then“about” as used herein indicates at least variations that may arise fromordinary methods of measuring and using such parameters. For example,“about” may comprise a variation of less than or equal to 5%, optionallyless than or equal to 4%, optionally less than or equal to 3%,optionally less than or equal to 2%, optionally less than or equal to1%, optionally less than or equal to 0.5%, and in certain aspects,optionally less than or equal to 0.1%.

In addition, disclosure of ranges includes disclosure of all values andfurther divided ranges within the entire range, including endpoints andsub-ranges given for the ranges.

Following severe trauma, the nervous system does not spontaneouslyregenerate, requiring intervention to restore function. There is a needto develop materials that enable the fabrication and implementation ofimproved and more effective nerve guidance scaffolds. In variousaspects, the present disclosure contemplates an improved and moreeffective tissue scaffold for promoting neural tissue growth andproliferation in a subject. The subject may be an animal with a complexnerve system, such as a mammal, like a human, primate, or companionanimal. The tissue scaffolds according to the present disclosure maythus be devices implanted in such a subject. As shown in FIG. 1, atissue scaffold 20 includes a sheath 30. Inside the sheath 30, aplurality of microchannels 40 are disposed. Each microchannel in FIG. 1thus includes a wall 42 and an open central lumen 44.

By “channel” it is meant that the structure defines an evidentlongitudinal axis and has an open lumen or hollow core. Channels havingsuch an evident longitudinal axis include an elongated axial dimension,which is longer than the other dimensions (e.g., diameter or width) ofthe channel. Thus, the elongated channels are linear. In certainaspects, such elongated channel has an aspect ratio (AR) defined as alength of the longest axis divided by diameter of the component, whichis preferably at least about 100 and in certain aspects greater thanabout 1,000. In yet other aspects, such channels may have an aspectratio of 10,000 or more.

The present disclosure thus contemplates a scaffold 20 comprising aplurality of microchannels 40 respectively defining a longitudinal majoraxis “L” as shown in FIG. 1. The term “micro-sized” or“micrometer-sized” as used herein is generally understood by those ofskill in the art to mean less than about 500 micrometers (μm) (i.e., 0.5mm). In accordance with certain variations of the present disclosure, a“microchannel” preferably has at least one spatial dimension that isless than about 1,000 μm. In certain aspects, each microchannel has aninner diameter of greater than or equal to about 10 μm to less than orequal to about 1,000 μm, optionally greater than or equal to about 10 μmto less than or equal to about 500 μm, optionally greater than or equalto about 50 μm to less than or equal to about 450 μm, optionally greaterthan or equal to about 50 μm to less than or equal to about 300 μm. Itshould be noted that so long as at least one dimension of themicrochannel falls within the above-described micro-sized scale (forexample, diameter), one or more other axes may well exceed themicro-size (for example, length and/or width). For example, depending onthe application, microchannels in accordance with certain variations ofthe present disclosure may have a length of greater than or equal toabout 500 μm to less than or equal to 30 cm, optionally greater than orequal to about 500 μm to less than or equal to about 10 cm, and incertain variations, optionally greater than or equal to about 500 μm toless than or equal to about 3 cm, by way of non-limiting example.

The microchannels are formed of a biocompatible and biodegradablematerial, such as a biocompatible polymer. By “biocompatible,” it ismeant that a material or combination of materials can be contacted withcells, tissue in vitro or in vivo, or used with mammals or otherorganisms and has acceptable toxicological properties for contact and/orbeneficial use with such cells, tissue, and/or animals. For example, abiocompatible material may be one that is suitable for implantation intoa subject without adverse consequences, for example, without substantialtoxicity or acute or chronic inflammatory response and/or acuterejection of the material by the immune system, for instance, via aT-cell response. It will be recognized that “biocompatibility” is arelative term, and some degree of inflammatory and/or immune response isto be expected even for materials that are highly compatible with livingtissue. However, non-biocompatible materials are typically thosematerials that are highly toxic, inflammatory and/or are acutelyrejected by the immune system, e.g., a non-biocompatible materialimplanted into a subject may provoke an immune response in the subjectthat is severe enough such that the rejection of the material by theimmune system cannot be adequately controlled, in some cases even withthe use of immunosuppressant drugs, and often can be of a degree suchthat the material must be removed from the subject. In certain aspects,biocompatible materials are those that are approved for use in humans byan appropriate regulatory agency, such as the Federal DrugAdministration (FDA) in the United States; the European Commission(EC)/European Medicines Agency (EMEA) in Europe; or Health Products andFood Branch (HPFB) in Canada.

For example, a scaffold structure can comprise microchannels formed frombiocompatible and biodegradable polymers, such as polyester polymers.Suitable biodegradable polymers for forming the microchannels include apolylactic acid, polycaprolactone (PCL), polyglycolic acid,poly(lactide-co-glycolide polymer (PLGA), and copolymers, derivatives,and mixtures thereof. In certain preferred aspects, the biocompatibleand biodegradable material is selected the group of polymers consistingof: polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), andcombinations thereof.

In certain aspects, the polymers can also be modified by chemical orphysical methods, such as cross-linking, heat treatment, photochemicaltreatment, and/or changes in the chemical or physical environment. Incertain aspects, the polymer modification occurs in a select portion orregion of one or more of the microchannels, or such polymer modificationcan occur to different degrees, potentially resulting in differentmaterials or material responses, as appreciated by one of skill in theart. Such polymer modification and/or treatment provide differentdegradation or release kinetics in certain aspects. Further, surfacealterations, such as differences in hydrophilicity, charge, or otherphysical properties, facilitate cell adhesion.

In certain aspects, the microchannels may be treated with abiofunctional agent or active ingredient; have different surfaceproperties or surface roughness; or have surfaces with differentmoieties exposed, which can be useful in designing spatially guidedcellular growth and in certain aspects to facilitate adhesion of cellsor tissue or to promote release of biofunctional agents, which includebiofunctional materials and active ingredients (e.g., pharmaceuticalactive ingredients), and the like, into the surrounding environment.

The biodegradable material forming the microchannel may dissolve,referring to physical disintegration, erosion, disruption and/ordissolution of a material and may include the resorption of suchmaterial by a living organism. In certain variations, biodegradablepolymeric material may dissolve or erode upon exposure to a solventcomprising a high concentration of water, such as blood, serum, growthor culture media, bodily fluids, saliva, and the like. Thus, uponimplantation, the material may dissolve or disintegrate into smallpieces. For structural scaffold members, the dissolution rate (e.g., arate at which the structural member is resorbed by surrounding cells)can be designed so that sufficient cellular growth occurs prior to thestructure dissolving or disintegrating via the resorption process. Invarious embodiments, the tissue scaffold device is designed to have adegradation time or dissolution rate that coincides with an amount oftime that permits adequate neural tissue regrowth through the scaffoldto a target tissue in the subject. Depending upon the subject and thetime needed for recuperation and regeneration of the tissue, by way ofnon-limiting example, the degradation time may be greater than or equalto about 1 month to less than or equal to about 3 years, greater than orequal to about 1 month to less than or equal to 1 year, and in certainvariations, greater than or equal to about 1 month to less than or equalto 6 months. In this manner, the cellular scaffold structure supportsand promotes cell growth, cell proliferation, cell differentiation, cellrepair, and/or cell regeneration in three-dimensions, especially forneural tissue growth.

In certain aspects, the walls 42 of the microchannels 40 are porous. Thepore size may be selected to promote substantially linear neural oraxonal tissue growth along the longitudinal axis “L” while avoiding cellgrowth through and across the microchannel walls 42. In certain aspects,the walls 42 are highly porous, for example, having a porosity ofgreater than about 1% to less than or equal to about 99%, optionallyhaving a porosity of greater than about 10% to less than or equal toabout 95%. The plurality of pores within the walls 42 may include aplurality of internal pores and external pores that are open to oneanother and form continuous flow paths or channels through the wall 42extending from a first internal surface 46 to a second external surface48. As used herein, the terms “pore” and “pores” refer to pores ofvarious sizes, including so-called “macropores” (pores greater than 50nm diameter), “mesopores” (pores having diameter between 2 nm and 50nm), and “micropores” (pores having diameter of less than 2 nm), wherethe pore size refers to an average or median value, including both theinternal and external pore diameter sizes.

The walls 42 of the microchannels 40 optionally comprise a plurality ofpores having an average pore size diameter of less than or equal toabout 50 μm, optionally less than or equal to about 40 μm, optionallyless than or equal to about 30 μm, optionally less than or equal toabout 20 μm, and in certain variations, optionally less than or equal toabout 10 μm. In certain aspects, the plurality of pores in themicrochannel 40 wall 42 has an average pore size that eliminatesline-of-site pores that could allow axons to grow between respectivemicrochannels 40. In certain other aspects, the average pore sizes inthe walls 42 may be macropores ranging from greater than or equal toabout 30 μm to less than or equal to about 50 μm. Such pore sizespromote flow of oxygen and nutrients through the walls 42 of themicrochannel 40 from the external surface 48 to the internal surface 46to support cells growing within the open central lumen 44, whileminimizing or preventing cells from being able to grow through themicrochannel walls 42. As will be discussed herein, techniques formaking the scaffolds 20 introduce porosity and surface roughness atlevels that promote cell adhesion to the microchannel 40 walls 42. Inthis manner, the scaffold 20 promotes cell growth, proliferation,differentiation, repair, and/or regeneration. In certain variations, thetissue is a neural tissue, such as axons.

Each microchannel 40 within the sheath 30 comprises a porous wall 42. Incertain aspects, suitable wall 42 thicknesses are the smallestthicknesses possible that retain structural integrity to the channel. Incertain aspects, the wall has a thickness of less than or equal to about500 μm. In other aspects, the wall has a thickness of less than or equalto about 100 μm. Where wall thicknesses are greater than 100 μm, theycan reduce the amount of space available within the open central lumen44 for axonal regeneration. In certain variations, the wall thicknessmay be greater than or equal to about 10 μm to less than or equal toabout 100 μm, optionally greater than or equal to about 10 μm to lessthan or equal to about 70 μm, optionally greater than or equal to about20 μm to less than or equal to about 70 μm, optionally greater than orequal to about 25 μm to less than or equal to about 67 μm, and incertain aspects, optionally greater than or equal to about 20 μm to lessthan or equal to about 50 μm. In certain other variations, the wall hasa thickness of greater than or equal to about 10 μm to less than orequal to about 20 μm.

One particular advantage of the tissue scaffold 20 design according tovarious aspects of the present disclosure is providing an overall openvolume (e.g., open lumen volume, including the volume of openinterstitial channels 56 within sheath and open central lumen 44 ofmicrochannels 40) of greater than or equal to about 50 volume %,optionally greater than or equal to about 60 volume %, optionallygreater than or equal to about 70 volume %, optionally greater than orequal to about 80 volume %, and in certain preferred aspects, optionallygreater than or equal to about 90 open volume % of the overall scaffold20 volume. It should be noted that conventional scaffold designs werenot able to achieve such high levels of open lumen volumes, which isbelieved to be particularly advantageous in supporting and promotinggrowth of healthy neural tissues having desirably high directionallinearity and high signal fidelity.

In certain aspects, a diameter “D” of each microchannel 40 of theplurality of microchannels disposed within the sheath 30 is selected tobe the same (or substantially the same accounting for small dimensionalvariances during manufacturing), although in alternative variations, thediameters D may intentionally vary between distinct microchannels 40 ofthe plurality present in the sheath (not shown in FIG. 1). As notedabove, in variations where the plurality of microchannels 40 havesubstantially the same diameter, an average inner diameter D isoptionally less than or equal to about 450 μm or any of the other rangesspecified previously. Each microchannel 40 may have an oval or sphericalcross-sectional shape to form microcylinder shapes that createsignificant open interstitial volumes in interstitial channels 56,although in alternative less preferred variations, other shapes may beused. Where the plurality of microchannels 40 has substantially the samediameters, they may be configured to be closely packed in an arraywithin the sheath 30. Thus, a portion of each microchannel 40 contactsanother adjacent microchannel 40. The plurality of microchannels 40 maybe arranged within the sheath 30 in a close-packed array that may createa honeycomb type of arrangement. In this manner, the tissue scaffolds ofthe present disclosure comprise discrete, linear, thin-walled,close-packed arrays of microchannels 40 disposed within externalprotective sheath 30. A microchannel density may be varied in differentembodiments, for example, the microchannel density may be greater thanor equal to about 1 to less than or equal to about 300 microchannels/mm²in the scaffold. In certain variations, the microchannel density may begreater than or equal to about 10 to less than or equal to about 30microchannels/mm². In another variation, the tissue scaffold may have amicrochannel density of about 120 microchannels/mm².

The sheath 30 may be formed of a biocompatible and/or biodegradablematerial that may be the same as or different from the microchannels 40.Desirably, the sheath 30 may have a similar porosity to themicrochannels 40 to promote flow and transport of nutrients to themicrochannels, while minimizing or preventing cellular growth from aninterior region 32 through the sheath to an exterior region 34. Thesheath 30 is shown as a cylindrical tube shape with an oval orcylindrical cross-sectional shape; however, the sheath 30 may have avariety of other shapes, so long as the microcylinders can be arrangedin an array within the sheath 30. Thus, in certain aspects, the sheath30 may have other shapes, including a butterfly shape similar to thatfound in a human spinal column by way of non-limiting example. Thesheath 30 may have a length that is the same as the microcylinders 40 ormay be longer for additional protection and securing to a portion of anerve 50 or surrounding tissue (e.g., by anastomosing). In this manner,the tissue scaffold 20, including the sheath 30 and microchannels 40 canextend over any distance to match injuries of individualsubjects/patients.

The scaffold 20 can be filled with cells. These cells can be modified toexpress a growth factor or can be therapeutic in nature such as stemcells or Schwann cells.

A portion of nerve 50, such as a nerve end, of the subject may bedamaged or severed, for example, a fully or partially lesioned nerve endcaused by injury, disease, or surgery. In certain aspects, a portion ofthe nerve end may be surgically divided, sectioned, cut, and/ortransected into one or more individual branches or fascicles that may besecured to a proximal end 52 or distal end 54 of the tissue scaffold 20.The one or more individual branches or fascicles of the nerve end 50 maycontact or be placed within one or more microchannels 40. The nerve end(or its individual branches or fascicles) can be secured via sutures,adhesives, or other known securing techniques to the proximal or distalends 52, 54 (shown in FIG. 1 as the distal end 54). Over a period of,for example, several months, the neural tissue originating from thenerve end 50 can grow along the longitudinal axis L of each microchannel40 and reinnervate any neural targets at the opposite end of the tissuescaffold 20. The tissue scaffolds according to various aspects of thepresent teachings thus facilitate neural tissue growth through the opencentral lumens 44 of the plurality of microchannels 40 from a first end(e.g., proximal end 52) to a second opposite end of the (e.g., distalend 54) scaffold 20. As will be appreciated by those of skill in theart, while the design of the inventive tissue scaffolds is particularlysuitable for promoting neural tissue growth, in alternative variations,the tissue scaffold may be used for other types of tissue growth.

In other aspects, surfaces of the walls 42 of microcylinders 40 may becoated with a biofunctional agent to promote cell growth, regeneration,differentiation, proliferation, and/or repair, for example. By“promoting” cell growth, cell proliferation, cell differentiation, cellrepair, or cell regeneration, it is meant that a detectable increaseoccurs in either a rate or a measurable outcome of such processes occursin the presence of the biofunctional agent as compared to a cell ororganism's process in the absence of such a biofunctional agent, forexample, conducting such processes naturally. By way of example, asappreciated by those of skill in the art promoting cell growth in thepresence of a biofunctional agent may increase a growth rate of targetcells or increase a total cell count of the target cells, when comparedto cell growth or cell count of the target cells in the absence of sucha biofunctional agent.

In certain variations, the biofunctional agent promotes cell growth,cell adhesion, cell proliferation, cell differentiation, cell repair,and/or cell regeneration by increasing a measurable process result(e.g., measuring total cell counts for cell generation or cellregeneration, measuring the rates or qualitative outcome of cellproliferation, cell differentiation, or cell repair rates) by greaterthan or equal to about 25% as compared to the result of the process inthe absence of the biofunctional agent, optionally increasing by greaterthan or equal to about 30%, optionally increasing by greater than orequal to about 35%, optionally increasing by greater than or equal toabout 40%, optionally increasing by greater than or equal to about 45%,optionally increasing by greater than or equal to about 50%, optionallyincreasing by greater than or equal to about 55%, optionally increasingby greater than or equal to about 60%, optionally increasing by greaterthan or equal to about 65%, optionally increasing by greater than orequal to about 70%, optionally increasing by greater than or equal toabout 75%, optionally increasing by greater than or equal to about 80%,optionally increasing by greater than or equal to about 85%, optionallyincreasing by greater than or equal to about 90%, and in certainaspects, optionally increasing by greater than or equal to about 95%.

Such a biofunctional agent may be introduced after the microcylinders 40are formed, for example, by coating, infusing, or otherwiseincorporating the biofunctional agent onto one of more surfaces (e.g.,internal surface 46) of the microchannel wall 42. In certain aspects, asurface of the porous wall 42 has a coating comprising a material forpromoting growth of the neural tissue selected from the group consistingof: fibronectin, keratin, laminin, collagen, and combinations andequivalents thereof. In certain variations, the walls may be coated withfibronectin, which has been found after screening over a dozen compoundsto be particularly advantageous with the biocompatible polymers formingthe microchannel walls to optimize cell and axon attachment.

The present technology thus enables a major advance over existingtechnologies in surgical repair of injured peripheral nerves. There arecurrently seven FDA-approved devices on the market for peripheral nerverepair. However, all of these existing devices consist of only a singleopen channel (not divided into individual microchannels) in which axonsfrequently diverge from linear paths, reducing the number of axons thatreach the distal end of the scaffold and contribute to nerve repair.Simpler designs like those commercially available more commonly resultin painful neuromas because of axon misguidance. Additionally, theproperties of materials out of which existing scaffolds have beenfabricated do not adequately support cell and axon attachment.Furthermore, many of the materials out of which conventional tissuescaffolds have been made, including hydrogels, have shown significantand problematic inflammatory response. Based on empirical observationafter implanting and testing hydrogel nerve regeneration scaffolds,hydrogel-based materials do not exhibit adequate strength to enable thefabrication of thin (<50 μm) wall scaffolds. Yet based on calculations,it appears that wall thicknesses of less than 50 microns are necessaryto achieve >90% lumen volume scaffolds that adequately support andpromote neural tissue growth. Thus, hydrogel based materials cannotprovide scaffolds having adequate strength with advantageous open lumenvolume provided by certain aspects of the present teachings.

The present tissue scaffold devices are superior in providing amulti-lumen design that enhances nerve guidance, thereby increasing thetotal number of axons that regenerate successfully. As a result, suchtissue scaffold devices work over long nerve gaps and after moreproximal nerve injuries, thereby addressing a great unmet medical need.Further, the tissue scaffolds according to the present disclosure aremade from biocompatible and biodegradable materials, such as PCL andPLGA polymers, with optimized porosity and surface roughness, providingsuperior cell adhesion levels and directional cell growth whileexhibiting significantly reduced inflammatory response in vivo afterimplantation. When tested in vivo, the devices of the present disclosureare biocompatible. Further, when directly compared side-to-side withcurrent FDA-approved scaffolds for peripheral nerve repair, theinventive tissue scaffold design is superior: a greater number of axonsare linearly organized and reach the distal end of the scaffold.

In this manner, the tissue scaffold devices according to certain aspectsof the present disclosure enable one or more of the following uniquefeatures or advantages: a close-packed array of linear microchannels(e.g., each microchannel having an inner diameter of ≥10 μm to ≤450 μm)that emulate native nerve organization as shown in FIG. 2A;microchannels having significant and customizable lengths; thin walledmicrochannels to maximize open volume (e.g., walls having a thickness of10-30 micrometers); high open lumen volumes (e.g., >90% in certainvariations); tissue scaffold devices comprising biocompatible materials,like FDA-approved polymer materials (e.g., PCL and PLGA); an ability tocontrol mechanical properties to optimize for strength to minimize wallthickness and suture-ability as an outer sheath tube; an ability tocontrol scaffold and sheath porosity to prevent axon penetration whileallowing permeation of oxygen and other nutrients; an ability to modifymicrochannel surface properties to enable cell attachment; a singleone-piece sheath and scaffold construction facilitating ease ofimplantation enabling secure apposition between nerve stumps andscaffold walls; and finally low material and fabrication cost. FIGS.2B-2C show cross-sectional schematics of two different high lumen volumenerve repair scaffolds which ideally match or emulate a nerve's nativearchitecture according to certain aspects of the present disclosure. Thedesign in FIG. 2C has a higher microchannel density than a microchanneldensity in FIG. 2B.

In accordance with other aspects of the present disclosure, a newmaterial processing technology is provided to enable the manufacturingof microchannel nerve guidance scaffolds with high lumen volumecomprising biocompatible polymer materials.

As noted above, many conventional nerve tissue scaffold devices areformed from hydrogels, which are too weak to form thin-walledmicrochannels. In replacing hydrogels, several FDA approved syntheticpolymers exhibit greater than 100 times in strength compared tohydrogels. However, these polymers also exhibit stiffnesses (elasticmodulus) that are significantly (approximately >100 MPa) higher thanhost nerve tissue (that is about 8 kPa), which could compromisebiocompatibility. Generally, it is believed that the nerve guidancescaffold material should be comparable to that of the host nerve tissueto minimize inflammation. Thus, in one aspect, the present technologyprovides an approach to reduce the stiffness of synthetic polymers toimprove biocompatibility of the tissue scaffold devices. However, it hasbeen surprisingly found that the tissue scaffolds of the presentdisclosure may have a relatively high modulus, but inflammatory responsewhen implanted remains desirably low showing good biocompatibility.

Additionally, it is believed that nerve regeneration scaffold walls mayrequire interconnected porosity to allow nutrients and oxygen topermeate laterally between microchannels and the scaffold periphery.Introducing porosity can also lower the elastic moduli. Conventionally,templating by using a porogen, in particle form, may be used to displacevolume in a polymer as it polymerizes/solidifies. Once polymerization iscomplete, the porogen/polymer construct is immersed in a solvent toselectively dissolve the porogen to create pores. However, conventionalporogen particles, such as sodium chloride (NaCl) particles haverelatively large particles sizes that produce pore diameters of greaterthan about 63 micrometers. Thus, use of such a conventional porogen sizewould not permit formation of thin scaffold walls (e.g. having athickness <50 microns) and would undesirably create line-of-site voidsthat axons could penetrate. Additionally, these relatively largepores/discontinuities would compromise the scaffold mechanicalintegrity.

In accordance with certain aspects of the present disclosure, a porogenis prepared by reducing particle size and then used to templateFDA-approved synthetic polymers for nerve repair. By reducing theporogen dimensions (e.g., to less than or equal to about 10 micrometersin certain variations), synthetic polymer scaffold walls comprisingnumerous interconnected, pores having a reduced average pore size (e.g.,less than or equal to about 10 micrometers) are created. The reductionin pore size thus serves to desirably eliminate line-of-site pores thatcould allow axons to grow between microchannels, while maintainingadequate mechanical strength of walls formed from the porous polymers.

The present disclosure provides in certain variations methods of makinga tissue scaffold for neural tissue growth. The method may compriseadmixing one or more porogens and a polymeric precursor solutiontogether. The ratio of the polymer to porogen determines the volume % ofthe polymer and can be selected based on the targeted porosity andmechanical properties. The polymeric precursor solution may include apolymeric precursor and a first solvent.

The porogen may have an average particle diameter or size of less thanor equal to about 40 μm, optionally less than or equal to about 30 μm,optionally less than or equal to about 20 μm, and in certain variations,optionally less than or equal to about 10 μm. The porogen is preferablya material that is brittle and soluble in a second solvent that does notdissolve in the first solvent/polymeric precursor solution. As a roughestimate, a ratio of bulk modulus to the shear modulus indicates theductile/brittle behavior of a solid. According to Pugh's criterion, acritical value for a transition from brittle to ductile behavior is1.75. Thus, to facilitate particle reduction via mechanical comminution,porogens may be selected as having Pugh ratios of less than 1.75. Incertain variations, the porogen is selected from a group consisting of:sodium chloride, calcium chloride, potassium chloride, sugars, andcombinations thereof. A sugar may be selected from the group consistingof: sucrose, maltose, lactose, fructose, glucose, galactose, andcombinations thereof.

NaCl is a particularly suitable porogen owing to its insolubility insolvents used to dissolve biocompatible polymers (such aspolycaprolactone (PCL) and polylactic co-glycolic acid (PLGA)) and itssolubility in water, which does not readily dissolve PCL or PLGA.

To reduce the porogen size, a mechanical comminution technique may beused to mill or pulverize porogen particles (such as NaCl). In certainvariations, the method may thus comprise reducing a particle size of aprecursor of the porogen by ball milling the precursor before admixingit with the polymeric solution. In other variations, a planetary ballmill may be used to mill the porogen to reduce powder particle size. Themixing may be for greater than or equal to about 1 minute to less thanor equal to several hours, for example, less than or equal to about 2hours, optionally less than or equal to about 1 hour. The speed of themixing may be conducted at 100 RPM to 400 RPM. In certain aspects, themixing may be conducted at 400 RPM for 30 minutes.

In FIG. 3, Step 1 a shows reducing the porogen particle size by usingplanetary ball milling. Planetary ball milling refers to a type of ballmilling process that employs a planetary rotation motion. Planetary ballmilling imparts significantly more kinetic energy to powder particles,as compared to conventional ball milling, thus it can achieve smallerparticles. However, other forms of milling may be used to reduceparticle size of the porogen, where appropriate. A Retsch PM 100planetary ball mill may be used with a 250 ml agate vial containingtwelve 9.1 mm diameter agate mill balls (Hann, Germany). In onevariation to obtain the desired average particle size (about 6micrometers), different ball milling conditions are tested: various vialrotational speeds ranging from 100 to 400 RPM and various milling timesranging from 1 to 60 minutes. Additionally, various combinations of resttimes between rotation directions (CW and CCW) are explored. The effectof rest time is studied, because it is known that resting allows thevial contents to cool for improved particle size reduction. In certainvariations, suitable although non-limiting ball milling conditions maybe 400 RPM for 30 minutes with no rest intervals and only CW rotation.

A reduced particle size porogen may thus be combined with the polymericprecursor solution. The polymeric precursor solution may compriseprecursors (such as monomers, comonomers, or oligomers) of any of thebiodegradable polymers discussed above, including polycaprolactone,poly(lactic-co-glycolic acid) polymer, or combinations thereof. Thesolution may further include a first solvent in which the polymerprecursor is soluble, such as chloroform, by way of non-limitingexample. In certain variations, the polymeric precursor solution maycomprise greater than or equal to 0.2% to less than or equal to about20% by mass or weight of the polymer precursor in the total solutionincluding the first solvent. In certain aspects, the polymeric precursorsolution may comprise greater than or equal to about 0.5 weight % toless than or equal to about 5 weight % of polycaprolactone in the firstsolvent. In other variations the polymeric precursor solution maycomprise greater than or equal to about 1 weight % to less than or equalto about 15 weight % of poly(lactic-co-glycolic acid) polymer in thefirst solvent.

As shown in Step 1 b, the polymeric precursor solution may be introducedinto the ball-milled porogen. Further mixing of the porogen with thepolymeric precursor solution may form a suspension. The mixing may befor greater than or equal to about 1 minute to less than or equal toseveral hours, for example, less than or equal to about 2 hours oroptionally less than or equal to about 1 hour. The speed of the mixingmay be conducted at 100 RPM to 400 RPM. In certain aspects, the mixingmay be conducted at 400 RPM for 30 minutes. In certain variations, thesuspension may comprise a porogen, such as sodium chloride, at greaterthan 0 volume % to less than or equal to about 80 volume % and either(i) greater than 0 volume % to less than or equal to about 80 volume %of polycaprolactone in the suspension; or (ii) greater than or equal toabout 0 volume % to less than or equal to about 95 volume % ofpoly(lactic-co-glycolic acid) polymer in the suspension.

Step 2 of FIG. 3 includes Steps 2 a-2 d where a tube is formed. In Step2 a, a template is contacted with the suspension to coat at least onesurface with the suspension. The template may have a rod or a fibershape selected to have a predetermined diameter corresponding to thedesired diameter of the coating and ultimately the microchannel formedthereon. The template may be solid or have a hollow core or internallumen. The template may comprise a metal selected from a groupconsisting of: brass alloy, copper, stainless steel, and combinationsthereof. It should be noted that certain template materials did notappear to work with certain material suspensions in not providingadequate stability or uniform wetting, for example, templates made ofgraphite, tungsten, and cross-linked polystyrene. Suitable metalmaterials for the template desirably fulfill the following criteria: (1)wettability by the suspension/slurry to produce a uniform thickness, (2)adequate stiffness to maintain linearity in high aspect ratio form and(3) delamination of the polymer after polymerization.

The template may have a diameter corresponding to the desired diameterof the microchannel to be formed (for example, for a solid template, theouter diameter of the template corresponds to the inner diameter of themicrochannel). Thus, any of the diameters discussed above in the contextof the microchannel may be appropriate diameters for the template. Thetemplate may be significantly longer than the desired length of themicrochannel and may be used to form multiple microchannels. Thecontacting may include immersing the template in the suspensioncomprising porogen and polymeric precursor to coat the surface with thesuspension.

As background on template material selection, while polymer opticalfiber templates can be used to fabricate greater than 60 volume %conventional nerve repair scaffolds, they are formed of hydrogels. Thesolvents used in the precursor solution (e.g., for PCL and PLGA) wouldreadily dissolve the polymer fibers if they are used as templates. Forexample, optical fiber templating typically are formed of noncross-linked thermoplastic polymers such as polystyrene orpolymethylmethacrylate (Paradigm Optics. Vancouver, Wash.). Because thesolvents used to synthesize PCL and PLGA are aggressive (chloroform),the polymer optical fiber templates would readily dissolve. Anothertemplate could be a sugar fiber comprising sucrose to create linearchannels in PLGA. Sugar is not soluble in the solvents that dissolvePLGA (or PCL). However, the close packing of sugar fibers has not beendemonstrated, thus, sugar fibers are unlikely to be able to form a >90%channel lumen volume. Furthermore, sugar is inherently brittle, thusprocessing high aspect ratio (about 200 micron diameter-1 cm long)fibers would likely be difficult to achieve.

As noted above, instead of using a solvent to remove fiber templates, ametal fiber or rod template is instead used. The template is combinedwith mechanical/chemical removal that makes high aspect ratiomicrochannels. In certain aspects, the suspension has adequate viscosityto allow the uniform coating of the template rods while maintaining theuniform dispersion of the porogen. The viscosity of the solution isdetermined by the concentration of the polymeric precursor solution(e.g., monomer solution), the solvents used, and the volume fraction andsize of the porogen included. The viscosity can be optimized by varyingone or all of these parameters.

After the template is removed from contact with the suspension, aportion of the first solvent may be removed from the coating formed onthe surface of the template as shown in Step 2 b. In this manner, theprecursor may be polymerized while having the porogen distributedtherein to form a coating. In certain aspects, the porogen may behomogenously distributed through the polymer in the coating. Notably,the coating may be formed on the outside of a solid template or on theinside and/or outside of a hollow template. The template may be rotatedor moved to facilitate volatilizing the first solvent from the coating.After a portion of the first solvent is removed, the coating thatremains on the template surface includes porogen and the polymer.

In certain aspects, the method may further comprise exposing the coatingto a lubricant or a delamination agent as shown in Step 2 c. Thelubricant/delamination agent may be a liquid that aids in delaminationand removal of the coating from the surface of the template. Thetemplate may be immersed in such a lubricant solution. A suitablelubricant includes methanol. Methanol does not dissolve the template,porogen, or polymer, but does cause the polymer to swell to ease andfacilitate delamination.

Next, the coating, comprising the porogen and the polymer, is removedfrom the template as shown in Step 2 d as a tube. The coating may beslid off the template.

After removal, in Step 2 e the tube/coating may be cut on one or moreends to form a microchannel. The cutting may be accomplished bymechanical cutting (e.g., by a razor or wire cutter), laser cutting, andthe like.

This process may be repeated to form a plurality of microchannels (or aplurality of microchannels may be made in a single process step and cutinto discrete microchannels). The microchannel is thus disposed withinand assembled inside a sheath with a plurality of other microchannels toform the tissue scaffold, as shown in FIG. 3, Step 3 a. The microchannelmay be introduced into the sheath or the plurality of microchannels maybe assembled and then the sheath formed over the assembledmicrochannels. The microchannel tubes may be stacked inside the sheathand then can be fused together. Thus, a process to assemble individualmicrochannels into close-packed arrays is contemplated. The use of sucha tube fabrication process can also fabricate relatively large outersheaths to contain and mechanically support the microchannel array.Additionally, such an outer sheath also assists in the suturing of thenerve stump to the scaffold.

Next, in Step 3 b, the microchannels and sheath (comprising the porogenand polymer) are exposed to a second solvent to remove the porogen fromthe materials. The second solvent dissolves the porogen, but not thepolymer. A suitable second solvent may be water. The construct may beimmersed in the second solvent, such as water, at ambient temperatureand pressure conditions. Excessive heating of the water bath may causedamage to the polymer and thus is desirably avoided. The duration ofexposure to the second solvent is sufficient to remove substantially allof the porogen to form a porous material. In certain aspects, exposureto the water may be greater than or equal to about 1 hour up to about 2days, optionally greater than or equal to about 2 hours up to about 1day, and in certain aspects, greater than or equal to about 3 hours upto about 18 hours. After removal of the porogen the tissue scaffold isformed that comprises a porous microchannel and/or porous sheath.

An additional step may be conducted that includes coating the tissuescaffold construct with a biofunctional material, as discussed above. Inthis manner, the scaffold can be coated with a desired protein ormolecule to improve biocompatibility and/or cell attachment.

The tissue scaffold may also be sterilized for implantation. Forexample, the scaffold may be immersed in ethanol for sterilization. Inother variations, the sterilization may be exposure to UV radiation orother known techniques that do not degrade or harm the polymericmaterials forming the scaffold.

To fabricate microchannel tubes in accordance with certain aspects ofthe present disclosure, a precursor of a polymer of interest isdissolved in a first solvent and ball milled with the pre-ball-milledporogen. A metal rod is then used as a template and can be coated withthe mixture. A polymeric tube is formed around the rod. The diameter ofthe rod determines the diameter of the tubes. The thickness of the tubeformed on the surface of the template rod is manipulated by changing theviscosity of the polymeric solution and the number of coatings. The rodis then removed from the suspension of porogen and polymeric precursorand a hollow polymeric tube is formed. An outer sheath with a largerdiameter, equivalent to the diameter of the implant, is fabricated usingthe same technique. The outer sheath can have a different materialcomposition than the inner microchannel tubes. The inner microchannelsand outer sheath are cut to a predetermined length depending upon theend use. The outer sheath can be larger (e.g., longer) than the innertubes if needed; for example, to provide extra support for the scaffoldand/or to have extra space to suture/staple the implant to the tissue.Microchannel tubes are then stacked inside an outer tube and can befused together. The scaffold is placed inside a solvent that etches awaythe porogen, but not the polymer. The scaffold may then be sterilized,for example, by using ultraviolet radiation or alcohol treatment.

Various embodiments of the inventive technology can be furtherunderstood by the specific examples contained herein. Specific Examplesare provided for illustrative purposes of how to make and use thecompositions, devices, and methods according to the present teachings.

EXAMPLE 1

Planetary ball milled porogen NaCl (6 micron diameter average particlesize) (Columbus Chemical Industries, INC. Columbus, Wis.)) is mixed withvarious concentrations of synthetic polymer, in chloroform (Alfa Aesar.Ward Hill, Mass.) (FIG. 3, step 1 b). PCL (Mn 80,000) (Sigma. St. Louis,Mo.) and PLGA (85/15) (PCAS. Longjumeau, France) are the polymersinvestigated in this example, thus various monomer concentrations areinvestigated and range between 0-10% by weight polymer dissolved insolvent (chloroform). Although a broad range of polymer concentrationscan be used, in this example, typically 3.0 wt. % PCL and 6.7 wt. % PLGAare used.

To control the volume % porosity, various volume concentrations of NaClare added to the monomer solutions ranging between 0% to 90%. To ensurethe slurries are well mixed and that the NaCl particles are homogenouslywetted by the monomer solution, they are planetary ball milled. Forexample, the mixture may be ball milled for 20 minutes under the sameball-milling conditions as the NaCl, discussed previously. The slurriesare cast into thin-wall tubes according to the process discussed aboveand shown in FIG. 3.

Such techniques are used to form thin-walled high lumen volume scaffoldsfor nerve repair. There are several approaches to fabricate open channelnerve growth scaffolds. A variety of metal fibers are used to fabricatechannels. A range of metal fibers (a fiber being analogous to rod orwire) are investigated and are selected based on the following criteria:(1) wettability by the slurry to produce uniform thickness, (2) adequatestiffness to maintain linearity in high aspect ratio form and (3)delamination of the polymer after polymerization. Examples of the metalfiber templates investigated are copper (McMaster. Aurora, Ohio),stainless steel (grade 304) (McMaster), brass (McMaster), tungsten(Goodfellow Corporation. Cambridge, England), and graphite (not a metal,but attempted to be used as a template) (Goodwinds LLC. Mount Vernon,Wash.). It is also determined that the diameter of the rod affectedwetting, which is likely a result of capillary forces associated withthe fiber radius of curvatures.

Based on this investigation, the following are selected for the varioustube geometries: copper fiber (300 micron outer diameter) is used tomake microchannels, stainless steel fiber is used to fabricate 1.6 mminner diameter (ID) outer sheaths, brass fiber is used to fabricate the6.8 mm ID outer sheath for pig scaffolds. The general tube fabricationprocess is described in FIG. 3, Steps 2 a-2 f, above.

The metal fiber is immersed in the slurry or suspension of porogen andpolymeric precursor (FIG. 3 step 2 a, for approximately 5 seconds). Ifnecessary, step 2 a can be repeated to achieve the desired wallthickness. The wetted metal fiber is removed from the slurry and ismanually spun clockwise and counter clockwise to uniformly evaporate thesolvent (FIG. 3 step 2 b, for about 30 seconds). To aid in delamination,the metal fiber/polymer and porogen construct is immersed in methanol(FIG. 3, step 2 c). The methanol does not dissolve the metal fiber,porogen salt or polymer, but does cause the polymer to swell to easedelamination. The tube is mechanically separated by pulling the tube offthe wire (FIG. 3, step 2 d). Lastly, the tube is cut to the desiredlength with a razor or laser (FIG. 3, step 2 e). Finally, the tube maybe immersed or soaked in water (for example, at ambient conditions) inFIG. 3, step 2 f.

Assembling the channels into scaffolds is described in FIG. 3, step 3 a.The microchannels are assembled into close-packed arrays and insertedinto the outer sheath. Because the microtubes have the same diameter,for a given scaffold design, they self-assemble into a hexagonal orhoneycomb arrangement to maximize the packing density.

After assembly, the construct is immersed in water (24 hours) toselectively remove the NaCl porogen contained in the walls of the tubes(microtubes and outer sheath) as shown in FIG. 3, step 3 b.

Subsequent immersion in ethanol sterilizes the scaffold forimplantation.

EXAMPLE 2

The fabrication of a PCL microchannel scaffold for implantation into apig sciatic nerve model is shown in FIGS. 4A-4D. As shown in FIG. 4A,the microchannels (15 mm long, 280 microns inner diameter (ID), 30micron wall thickness) are assembled in a custom fabricated clam shell,slotted die fixture (FIGS. 4C-4D). The microchannels are stacked andcompressed to create an ordered array of microchannels. Once stacked, anouter sheath (6.8 mm ID) shown in FIG. 4B is inserted between themicrochannel array periphery and inner lining of the clam shell fixture.60 micro channels are packed into the 6.8 mm ID outer sheath creating a90% open lumen volume scaffold. A microchannel density of 120microchannels/mm² is formed with this scaffold. See FIGS. 5A-5C. Thisscaffold is implanted in a pig sciatic nerve model for in vivo andfunctional recovery testing.

Microstructural Analysis

To characterize the effect the porogen had on porosity, scanningelectron microscopy (SEM) is conducted. In FIG. 6, the fracture surfaceof a typical PCL and NaCl construct before salt leaching (immersion inwater to selectively remove NaCl) is shown. After planetary ballmilling, the average NaCl particle size is 17 microns. FIG. 6 also showsthe NaCl dispersed in the PCL.

FIGS. 7A-7B compare pure PCL polymer (no NaCl porogen in FIG. 7A) ascompared to salt-templated PCL in FIG. 7B. FIG. 7B shows how the NaClporogen creates porosity. Although not described in detail, there isevidence to suggest that when the NaCl is below a certain diameter(believed to be somewhere between about 17 and about 50 microns) and arelatively high volume fraction of NaCl (approximately >50%), theporogen disrupts PCL polymerization to create unique porosityunobtainable with the previous state-of-the-art salt leaching processes.

Such a salt leaching process can also be used with other FDA approvedbiocompatible polymers, such as polylactic co-glycolic acid (PLGA).Porous PLGA is thus formed. Essentially, the process described above inthe context of FIG. 3 is used, except an 85/15 (lactic acid/glycolicacid wt. % ratio) replaced the PCL. As shown in FIGS. 8A-8D, the samescale and volume fraction porosity is achieved in PLGA. FIG. 8A shows 0%porosity, FIG. 8B shows 40% porosity, FIG. 8C shows 50% porosity, andFIG. 8D shows 60% porosity. In the context of nerve repair, if the PCLdegradation rate is too slow for certain applications, PLGA is a viablealternative owing to its faster degradation rate.

By controlling the volume fraction of porosity, the following aspectscan be tuned: elastic modulus, strain to failure, strength, degradationrate, and cross-flow of nutrients between microchannels. Preliminarydata demonstrate the correlation between the volume fraction of porosity(or PCL volume fraction which is equal to 1 vol. % porosity) and theelastic modulus (stiffness) in NaCl-templated PCL (FIG. 9). From FIG. 9,it can be seen that the elastic modulus is sensitive to the volumefraction porosity over a broad range. For example, the elastic modulifor the 20 and 100 vol. % PCL are 1.1 MPa and 182.1 MPa, respectively.The elastic modulus value of 1.1 MPa is within a factor of four comparedto the 3 wt. % agarose (0.3 MPa) that has been used previously and iscompatible with nerve tissue. Additionally, the value of 182.1 MPa for100% PCL is in agreement with literature values for pure PCL.

The scaffolds have been designed, fabricated, and implanted in vivo inrodents, and pigs. Compared to state-of-the-art (SOA) single-lumendevices (such as commercially available INTEGRA® and NEUROLAC® scaffolddevices), the sheath and scaffold technology according to the presentteachings enable linear axon guidance and distal axonal penetration intohost tissue. Below are three examples of scaffolds designed andfabricated with in vivo efficacy testing: (1) a salt-templated PCLsheath combined with a previous generation hydrogel microchannelscaffold in the PNS; (2) a salt-templated PCL sheath and a microchannelscaffold prepared according to certain aspects of the present disclosurein the PNS; and (3) a salt-templated sheath and a microchannel scaffoldprepared according to certain aspects of the present disclosureimplanted in the CNS.

In Vivo Efficacy Testing for Salt-Templated PCL Sheath with AgaroseMicrochannels

To demonstrate the utility of salt-templated PCL sheaths in the PNS, 1.1mm ID, 1.1 cm long 30 vol. % PCL sheaths are fabricated (70 vol. %porosity) according to certain aspects of the present disclosure asshown in FIG. 10C. 1 cm long agarose microchannel scaffolds aremicro-drilled consisting of linear, 300-micron inner diameter channels(FIGS. 10A-10B). The agarose scaffolds are inserted into PCL sheaths andimplanted into Fischer rat sciatic nerves by an orthopedic surgeon. ThePCL sheaths are sutured to the sciatic nerve stumps on the proximal anddistal ends. After eight weeks in vivo, macroscopic analysis shows theintegration of the nerve stumps to the PCL sheaths is maintained, thePCL sheath remained intact, and the sutures are still in place (FIG.11). Eight weeks post-implantation, histological analysis indicatedrobust axon ingress and egress is achieved over the 1 cm longmicrochannels, as shown in images at the proximal and distal ends of thePCL sheath in FIGS. 12A-12B, In addition, robust integration of Schwanncells with regenerated axons is apparent.

Previously, chitosan-based sheaths in rat and pig sciatic nerve modelswere studied. In all cases, and despite the use of the low endotoxingrade, significant inflammation occurred. For example, a firstgeneration hybrid chitosan-sheath and agarose micro-channel scaffold wasimplanted in a pig (FIGS. 13A-13C). The scaffold/lesion cavity wasencapsulated in inflammatory tissue due to the presence of the chitosan.Thus, in comparison, the salt-templated PCL sheaths according to certainaspects of the present disclosure appear to be superior to commercialproducts based on chitosan such as the single-lumen product produced byMEDOVENT® (GmbH).

Salt-Templated PCL Sheath and PCL Microchannel Scaffold in the PNS

To demonstrate the utility of salt-templated PCL sheaths and PCLmicrochannel scaffolds prepared in accordance with certain aspects ofthe present teachings in the PNS, 1.1 mm ID, 1.1 cm long PCL sheaths arefabricated (30 vol. % PCL, 70 vol. % porosity). Several 1 cm long,300-micron inner diameter salt templated microchannel scaffolds arefabricated using the process outlined in FIG. 3 and as discussedpreviously. The scaffolds are implanted into Sprague-Dawley rat sciaticnerves by an orthopedic surgeon. The PCL sheaths are sutured to thesciatic nerve stumps on the proximal and distal ends. Eight weekspost-implantation, histological analysis indicated robust axon ingressand egress is achieved over the 1 cm long microchannels (FIGS. 14A-14C).In addition, robust integration of Schwann cells with regenerated axonsis apparent.

Salt-Templated PCL Sheath and PCL Microchannel Scaffold in the CNS

To demonstrate the utility of salt-templated PCL sheaths and PCLmicrochannel scaffolds prepared according to certain aspects of thepresent disclosure in the CNS, 1.8 mm ID, 2 mm long PCL sheaths arefabricated (30 vol. % PCL, 70 vol. % porosity). Several 2 mm long,260-micron inner diameter salt templated tubes are inserted into the 1.8mm ID tube (FIG. 15). The scaffolds are implanted into Fisher 344 femalerat T3 full transection lesion cavities. Four weeks post-implantation,histological analysis indicated the PCL scaffolds remained intact andthat, despite the addition of growth factors, there is clear evidence oflinear axon guidance and egress (yellow arrows in FIG. 16).

FIGS. 17A-17C show cell attachment on a control and PCL materials forpurposes of comparison. 3T3 fibroblast cells are stained for actin andnucleus. FIG. 17A shows a positive control of cell growth in a wellplate. FIG. 17B shows cell growth on non-porous PCL (100% by volumePCL). FIG. 17C shows 3T3 fibroblast cell growth on a porous PCL preparedin accordance with certain aspects of the present disclosure having 30volume % PCL (70 volume % porosity). The cells do not attach to thenon-porous PCL film in FIG. 17B, but attach to the porous PCL in FIG.17C. While not limiting to any particular theory, this may beattributable to the improvement in wettability of the porous PCLmaterial.

FIGS. 18A-18C show that the walls of microchannels formed haveinterconnected porosity, which is important for transportation ofnutrition, waste, and oxygen from cells contained inside the scaffold.FIG. 18A shows an SEM of the surface of the inner wall, FIG. 18B an SEMof the cross-section of the wall, and FIG. 18C the outer surface of thewall of the microchannel.

FIGS. 19A and 19B show two PCL tissue scaffolds made in accordance withcertain aspects of the present disclosure both having over 85% openvolume. FIG. 19A is rat spinal cord tissue scaffold (scale bar is 300μm), while FIG. 19B is a pig sciatic nerve scaffold (scale bar is 4 mm).

EXAMPLE 3

Two types of microchannel tubes are prepared; microchannels to guidenerves and large diameter sleeves to serve as the sheath that houses themicrochannel scaffolds and enables suturing of nerve stumps to thescaffolds. For a rat model scaffold, a stainless steel rod with adiameter of 1.6 mm is used. Copper wires with diameters of 300 μm areused to fabricate the inner tubes. For a pig PNS scaffold, a stainlesssteel rod with 6.8 mm diameter and brass wires with a diameter of 500 μmare used to fabricate the outer sleeve and the inner tubes,respectively. The rod or wire is placed in a suspension comprising 70vol. % of salt porogen and PCL or PLGA in solution. The rod or wire isthen quickly removed and spun while holding the rod/wire horizontally todry off the solvent. For pig scaffold inner tubes and outer sleeves,this process is repeated one more time. The polymer-coated wire/mold isthen placed in methanol and the polymeric tube is gently extracted. Thetubes are then cut to 1 cm for rat model and 1.5 cm for pig model. Sincethe tubes generally clamp together after being cut, a copper wire of 200μm in diameter is passed through the tubes to open them (thus ensuringpatency of the inner central lumen).

In substantial work done with transected rat sciatic nerve model,scaffolds containing linear guidance channels with diameters of 200 μmhave been observed to support highly linear regeneration of injuredperipheral axons. FIGS. 20A(1) and 20A(2) show distal and proximal endsof a multichannel 10 mm PCL scaffold prepared in accordance with certainaspects of the present disclosure in transected rat sciatic nerve, 4weeks post implant. Axons are labeled in red using NEUROFILLAMENT™ 200stain. Arrow heads point where the proximal transected nerve stump isanastomosed to the scaffold. Full arrows point to linear axons in thechannels on the proximal and distal parts of the scaffold as well as inthe egress. As shown in FIG. 20A (including FIGS. 20A(1)-20A(4)) a 10 mmpolycaprolactone (PCL) implant has many packed linear axons in theproximal as well as the distal part of the scaffold. These axonsacquired and kept a linear trajectory as they exit and continue throughthe distal nerve stump (FIG. 20A(1)).

However, as shown in FIGS. 20A(3)-20A(4), a comparative conventionalNEUROGEN™ open tube scaffold (sold by Integra LifeSciences) 4 weeksafter implantation in the rat transected sciatic nerve has many axonslosing linear orientation even on the proximal side, merely 200 μm afterthey enter the scaffold. FIG. 20A(4) (full arrows and circle where axonsare perpendicular to regeneration axis). The axons are less dense andthe few that reach the distal side are not oriented even if they exit tothe egress into the distal nerve. FIG. 20A(3). This misguidance of axonscan cause pain due to neuroma.

A 15 mm implant prepared in accordance with certain aspects of thepresent disclosure showed good integration with the nerve stumps as well(FIG. 20B). The scaffold is loaded with BDNF secreting-marrow stromalcells in order to augment the number of axons entering the scaffold. Inaddition, distal nerve injection of BDNF is performed to attractregenerating axons to exit the scaffold into the distal nerve. Theresult is extensive emergence of regenerating axons from the scaffold inBDNF treated animals (FIGS. 20C(1) and 20C(2) showing proximal anddistal sides. A cross-section of the microchannels shows many myelinatedaxons in the BDNF treatment, similar to an autograft as well asvascularization (FIGS. 20D(1) and 20D(2) showing the BDNF cross-sectionand syngeneic graft cross-section, respectively). Quantification of axondensity 12 mm within the scaffold indicates that in BDNF group axondensity in channel core is similar to syngeneic nerve autografts. FIG.20E. These successful results in a rat led to the scale up of thescaffold technology in the porcine model.

To further improve certain variations of the scaffold implant, a sheathdesign is used that envelopes the scaffold and extends 1.5 mm over theedges of the scaffold (FIGS. 21A-21C). The scaffold may be anastomosedto the epineurium without suturing directly to the scaffold and thusdamaging the structure. It also simplified the implantation procedurefor the surgeon, as it is becomes a common anastomosis as being done inhuman patients with predicated devices that are hollow tubes.

After complete transection of the porcine sciatic nerve 5 cm rostral tothe lateral femoral epicondyle, a 15 mm-long PCL scaffold prepared inaccordance with certain aspects of the present disclosure is implantedinto the injury site. When examined 3 months later, scaffolds are foundto have integrated efficiently. The scaffolds support linear axonalregeneration over the gap (FIGS. 22A-22B and FIGS. 23A-23D); axons areobserved in the distal stump of the nerve, 3 mm beyond the scaffold(FIG. 23D). Several regenerating axons became remyelinated by Schwanncells (FIG. 23D). Some sensory function is restored to the skin of thelateral front part of the leg (FIGS. 24A-24C). A tester applies pressureto the skin on different parts of the leg (arrows) and looks for limbretraction away from the stimulus. In FIGS. 24A(1)-24A(3) just aftertransection of the sciatic nerve, the pig's leg did not move. FIGS.24B(1)-24B(2) show preliminary results 3 months post scaffold implant.The testing detected sensitivity in the lateral front part of the leg(FIG. 24B(1), arrow). There is a prompt withdrawal of the limb as shownin FIG. 24B(2) indicating recovery of sensory function. FIG. 24C showsthe area on the skin is innervated by the Common fibular nerve—a branchof the sciatic nerve.

FIG. 25A is a schematic of the anatomical structures present in arepresentative peripheral nerve structure. FIG. 25B shows across-sectional view of a terminal end of a biomimetic micro-scaffoldprepared in accordance with certain aspects of the present disclosuredesigned to mimic the natural architecture of a peripheral nerve. FIG.25C shows comparative commercially available, Federal DrugAdministration (FDA)-approved hollow tube scaffold devices from left toright: NEURAGEN™ nerve guide available from Integra Lifesciences Corp.,NEUROTUBE™ available from Synovis Microcompanies Alliance, and NEUROLAC™available from Polyganics. The microchannel scaffold (MCS) device likethat shown in FIG. 25B is a technology that facilitates and preciselyguides axons across even long peripheral nerve injury gaps. Thebiomimetic micro-scaffolds prepared in accordance with certain aspectsof the present disclosure provide long gap (greater than about 1 cm)peripheral nerve guidance. Guidance is achieved by fabricating deviceswith precise linear channels to maintain the intact topography of theperipheral nerve with a far greater degree of fidelity than existing,single lumen channels like those shown in FIG. 25C. Thus biomimeticmicro-scaffold prepared in accordance with certain aspects of thepresent disclosure enables a device that micro-aligns specific nervebundles and guide regenerating axons directly to their respective targetfrom proximal to distal side of the injury mimicking the natural nervearchitecture like that shown in FIG. 25A.

Other advantages of the microchannel scaffold technology according tocertain aspects of the present disclosure are the unique materials andmaterials-processing techniques that produce devices that may have: 1)greater than about 60% microchannel open volume or any of the valuespreviously discussed above, 2) high number of microchannels per unitarea (for example, about 10-30 microchannels/mm²), 3) microchanneldiameters in the range of about 200 micrometers, 4) wall thicknesses ofgreater than or equal to about 25 to less than or equal to about 67micrometers, and 5) biodegradable properties. The biomimeticmicro-scaffold prepared in accordance with certain aspects of thepresent disclosures have succeeded in orienting and guiding in vivoaxonal regeneration in the spinal cord. No evidence of significantinflammation was observed in in vivo tests. Further, the biomimeticmicro-scaffold prepared in accordance with certain aspects of thepresent disclosure have been used to successfully repair nerve gapsafter peripheral nerve injury in rats and pigs.

EXAMPLE 4

A microchannel scaffold prepared according to certain aspects of thepresent disclosure formed of polycaprolactone (PCL), containing linearguidance channels about 200 μm in inner diameter, is used with atransected rat sciatic nerve model. The microchannel scaffold device cansupport highly linear regeneration of injured peripheral axons. FIGS.26A-26C show comparative growth in transected rat sciatic nerves 4 weeksafter implantation. FIG. 26A shows a comparative NEURAGEN™ scaffold,FIG. 26B shows a 10 mm inventive micro-channel scaffold, and FIG. 26Cshows an autograft. Green-NF200 stain is used for axons. The interruptedlines demarcate the implant-nerve interface. Excellent integration ofthe microchannel scaffold device is observed, similar to the predicatedevice NEURAGEN™ and to an autograft. The arrows on the left point tothe proximal side of the implant. A clear reduction in axon density isobserved in NEURAGEN™ device (FIG. 26A) while the microchannel scaffolddevice has similar axon density to the autograft (FIG. 26B). The arrowson the right point to the distal aspect of the implant. Almost no axonsreached to the distal part of NEURAGEN™ device, while the inventivemicrochannel scaffold device kept the same axon density as the autograft(FIG. 26C).

Unlike the comparative scaffold device NEURAGEN™, the inventivemicrochannel scaffold technology guides axons throughout the gap fromproximal to distal while maintaining axonal linearity (FIG. 26B and FIG.27)). In addition, densely packed and linear axons are detected in thedistal nerve and 3 mm beyond the lesion, similarly to the autograft(FIGS. 26A, 26C, and 27). Conversely, the NEURAGEN™ device exhibits amarked reduction in axon density and misalignment within the first fewmm of the proximal nerve end, potentially causing neuromas and pain. Forexample, in the NEURAGEN™ device, axons are already misguided at theproximal side and even turning back (arrows shown in the proximalscaffold). Axons are sparsely positioned on the distal nerve and 3 mmbeyond the lesion (arrows in the end of block).

Quantifying the axon number in the three groups supports the observationthat the number of regenerating axons is reduced dramatically, fromproximal to the distal part of the implant in the comparative NEURAGEN™device. In comparison, the axon guidance and density using themicrochannel scaffold technology according to certain aspects of thepresent disclosure is comparable to that observed in the autograft (FIG.28). The outer sheath of the inventive microchannel scaffold device alsocomprises PCL, which is mechanically robust and durable to allowhandling and suturing to the nerve stumps. This allowed anastamosis tothe epineurium, thus simplifying the surgery as it is becomes a commonanastomosis as being done in human patients with commercially availabledevices that are hollow tube, such as the comparative NEURAGEN™scaffold.

In some experiments, rat survival is extended for 11 weeks postimplantation. Rats are tested for sensory recovery. Nociceptivesensitivity (cutaneous innervation) is measured by applying a pinprickto the distal skin territory of the injured sciatic nerve (lateral hindpaw). A score of 2 indicates a normal response and complete recovery. Asa control, rats are tested on the medial side of the foot, innervated bythe saphenous nerve, to confirm normal function. Pin Prick analysisreveals no sensory recovery for 6 weeks in either sciatic nerveimplanted groups (score of 0; FIG. 29). However, rats with porous PCLscaffolds prepared in accordance with certain aspects of the presentdisclosure recover some nociceptive sensitivity at 7 weeks, whereas therats with comparative NeuraGen® guides recover some nociceptivesensitivity later, at 8 weeks. Sensory recovery is accelerated in thePCL-porous group compared to NeuraGen® guide. These findings stronglysupport accelerated axon growth in the inventive porous PCL groupcompared with the comparative NeuraGen® devices. Thus, microchannelscaffold devices prepared in accordance with the present disclosureprovide a greater potential for functional recovery and an overallimproved approach over current standard of care in treating large gapperipheral nerve injury.

EXAMPLE 5

Microchannel scaffold devices prepared in accordance with certainaspects of the present disclosure are further tested in a large animalmodel—the porcine model. After complete transection of the porcinesciatic nerve 5 cm rostral to the lateral femoral epicondyle, a 15mm-long PCL device is implanted into the injury site. When examined 4months later, the device integrated well with host tissue (FIGS.30A-30D) and supported linear axonal regeneration over the gap; axonsare observed in the distal stump of the nerve, 3.5 mm beyond the implantsite (FIG. 30D). In FIG. 30A, arrows point to the suture where thedevice is anastomosed with the epineurium of the nerve stumps. FIGS.30B-30C show axon labeling (NF200—green). In FIG. 30B, axonalpenetration occurs into the device on the proximal side. These reach thedistal side (FIG. 30C) of the device in a linear fashion. Theinterrupted lines demarcate host-scaffold interface. FIG. 30D showsassociated axons and Schwann cells observed in the distal stump, 3.5 mmbeyond the distal end of the implanted device. Thus, severalregenerating axons are remyelinated by Schwann cells.

Reinnervation of sensory nerve tracts is observed upon stimulus of theskin on the lateral front part of the leg, much like the test resultsshown in FIGS. 24A-24B at three months. The pig is also capable ofsupporting itself on the leg with the implant. The hoof is deformed asthe pig shifted its weight balance in the first few days to recovery, soit supports the leg with the implant on the hoof itself and not on thetoes. This position is not changed even as the pig is recovering andfunctional regeneration regained. This procedure has been successfullycompleted in three pigs with similar results, including demonstratingsensory regeneration.

As such, the inventive technology can be used to achieve peripheralnerve repair after traumatic injury. The new tissue scaffold devicesexhibit superiority to existing FDA-approved devices for treating longergap injuries and more proximal, large gap injuries. When implanted in asubject, such tissue scaffolds have low levels of inflammation andimmune system response in contrast with conventional devices. Inaddition, the inventive technology can be individualized to match theinjury in each patient, among other advantages.

The foregoing description of the embodiments has been provided forpurposes of illustration and description. It is not intended to beexhaustive or to limit the disclosure. Individual elements or featuresof a particular embodiment are generally not limited to that particularembodiment, but, where applicable, are interchangeable and can be usedin a selected embodiment, even if not specifically shown or described.The same may also be varied in many ways. Such variations are not to beregarded as a departure from the disclosure, and all such modificationsare intended to be included within the scope of the disclosure.

What is claimed is:
 1. A method of making a tissue scaffold for neuraltissue growth, the method comprising: admixing (i) a porogen having anaverage particle size of less than or equal to about 40 μm with (ii) apolymeric precursor solution comprising a biocompatible andbiodegradable polyester polymer precursor and a first solvent, to form asuspension; contacting a template with the suspension to coat at leastone surface of the template; volatilizing at least a portion of thefirst solvent to form a coating; removing the coating from the template;to create a microchannel; assembling the microchannel inside a sheathwith a plurality of other microchannels; and removing the porogen toform a plurality of porous microchannels disposed within sheath thuscreating the tissue scaffold.
 2. The method of claim 1, wherein themethod further comprises cutting the coating to form a microchannelprior to the assembling.
 3. The method of claim 1, wherein thebiocompatible and biodegradable polyester polymer is selected from thegroup consisting of: polycaprolactone, poly(lactic-co-glycolic acid)polymer, and combinations thereof.
 4. The method of claim 1, wherein theporogen is a material that is brittle and soluble in a second solvent,but does not dissolve in the first solvent.
 5. The method of claim 4,wherein the porogen is selected from a group consisting of: sodiumchloride, calcium chloride, potassium chloride, sugars, and combinationsthereof.
 6. The method of claim 1, further comprising ball milling theporogen before the admixing.
 7. The method of claim 1, wherein theadmixing comprises ball milling the porogen, the polyester polymerprecursor, and the solvent together for a duration of greater than orequal to about 1 minute to less than or equal to about 60 minutes. 8.The method of claim 1, wherein the template has a rod or a fiber shapeand comprises a metal selected from a group consisting of: brass alloy,copper, stainless steel, and combinations thereof.
 9. The method ofclaim 1, further comprising coating the surface of the porous coatingwith a biofunctional material for promoting growth of the neural tissueselected from the group consisting of: fibronectin, keratin, laminin,collagen, and combinations thereof.
 10. The method of claim 1, furthercomprising exposing the porous coating with a delamination agent tofacilitate the removing of the coating from the template.
 11. The methodof claim 1, wherein the polymeric precursor solution comprises: (i)greater than or equal to about 0.5 weight % to less than or equal toabout 5 weight % of polycaprolactone in the first solvent; or (ii)greater than or equal to about 1 weight % to less than or equal to about15 weight % of poly(lactic-co-glycolic acid) polymer in the firstsolvent.
 12. The method of claim 1, wherein the suspension comprises aporogen comprising sodium chloride at greater than 0 volume % to lessthan or equal to about 80 volume % and either: (i) greater than 0 volume% to less than or equal to about 80 volume % of polycaprolactone in thesuspension; or (ii) greater than or equal to about 0 volume % to lessthan or equal to about 95 volume % of poly(lactic-co-glycolic acid)polymer in the suspension.